Various spectroscopic methods and instruments have existed for years that can be used to perform chemical analysis and identification of materials. Such chemical analysis methods and instruments utilize, for example. Raman spectroscopy, and/or fluorescence spectroscopy phosphorescence spectroscopy. These methods and instruments have been used in a wide range of biological and chemical research, as well as clinical, industrial, and governmental applications. These methods and instruments are being increasingly used in commercial and governmental applications such as for product inspection during the manufacture of pharmaceutical and medical products, manufactured food and chemical products, environmental testing, hazardous material detection, and other applications.
When a sample is exposed to radiation (e.g. infrared (IR) radiation, visible light, or ultraviolet (UV) radiation) at a given frequency, some of the radiation may be transmitted through the sample. Some of the radiation may be elastically scattered and retains the same frequency as the incident radiation. Some of the radiation may be absorbed in the sample. The absorbed radiation is either re-emitted after interaction with the sample or converted to thermal energy in the sample. The re-emitted radiation is sometimes referred to as inelastically scattered radiation. The inelastically scattered radiation is re-emitted as fluorescence or phosphorescence at wavelengths longer than, or frequencies lower than, the irradiation or excitation frequency, and a small fraction may be re-emitted as Raman scattered radiation. Fluorescence or phosphorescence emissions are red shifted from the excitation frequency and have a spectral distribution that is relatively independent of the excitation frequency. Raman emissions are dependent on excitation frequency and are measured as a sum or difference frequency from the excitation frequency. Absorption of radiation requires that the energy of the exciting photon be of higher energy than that of an excited state of the molecule that is being targeted. Raman emissions can be either blue (anti-Stokes) or red (Stokes) shifted from the excitation frequency by an amount determined by the rotational and vibrational bonds within the molecules being irradiated. Raman scattering efficiency is typically very low compared to fluorescence. However, when the energy of the excitation radiation corresponds to strong absorption bands of an analyte, a resonance effect can amplify the Raman signal by many orders of magnitude. Another detection method in common use uses infrared radiation and is known as Fourier Transform Infrared (FTIR) spectroscopy.
Detection and identification of materials may be one objective but when identifying trace amounts of a material on a surface another useful or even more important parameter may be the quantification of such trace material at different locations on the surface (e.g. as a surface density of the material at a given location, for example in units of nanograms (ng) or micrograms (ug) per square centimeter (cm2)). Quantification may be the sole parameter of interest in situations where identify of the trace material is already known.
Traditional methods for identifying and quantifying trace chemical or biological contamination on surfaces employs:                (1) swabbing a defined surface area,        (2) removing the chemical or biological material from the swab using sonication in a liquid or similar process, and        (3) examining the liquid with, for example, high performance liquid chromatography, capillary electrophoresis, mass spectrometry, or other methods which may or may not be electromagnetic spectrographic methods.This process and variants have been employed for many decades as the standard by which chemical or biological contamination on surfaces has been determined. This process is regulated by the U.S Food and Drug Administration, and other government agencies as the gold standard for identifying and quantifying such trace materials. This process is long and tedious, takes significant manpower and equipment, and has significant short comings resulting from inherent process limitations and implementation limitations. Such flaws include inaccuracies due to losses of sample during extraction, losses during sonication, transfer to an analytical method, and the like, which causes the ultimate concentration determination to be flawed. This process is used in cleaning validation in the pharmaceutical, food, chemical, biological manufacturing businesses as well as other places where contamination of surfaces is of importance and is regulated by government or internal standards.        
FIG. 1 illustrates a more detailed process flow according to an example of prior art cleaning validation processes. The process 100 starts with block 101 where it is assumed that cleaning has occurred, and that validation is to be undertaken. The process then flows to block 102 which calls for preparation of a validation cart that will be used in block 103 to move all of the materials and any necessary apparatus to the room or equipment that is to be validated. From block 103 the process moves to block 105 which calls for the use of a plurality of swabs to collect samples from defined areas to be examined. From block 105 the process moves to block 106 which calls for taking the swabs to a laboratory while 107 calls for setting up the laboratory. From block 107 the process moves to block 108 which calls for extracting the samples from the plurality of swabs while carefully controlling the handling of the swabs, or at least samples from swabs, such that associated sampling locations and samples remain correlated. Once appropriate samples have been extracted, the samples are run through HPLC analysis according to block 109 which is followed by data evaluation in block 110 and disposal of the consumed sample material in block 111. Finally, in block 112 the report on cleanliness validation is ready which may be 24, 48, or even 72 hours, or more, after the samples were originally taken. In block 113, a determination is made as to whether cleanliness requirements were met for each location, and if they have been, the process ends at block 116, but if they have not, the process moves to block 114 wherein a global or local decision on recleaning is made and implemented for at least some locations after which the process loops back into block 102 for another loop through the cleaning validation process. Of course, in the prior art, variation of this process is possible but the main take away is that the standard process in use today is manpower intensive, takes a long time, and has a slow feedback loop that limits how quickly cleaning is validated, recleaning can be performed, and the equipment or room put back into use for product production.
Significant efforts have been taken recently to update technology used in detecting concentrations on surfaces in many different application areas including cleaning validation in pharmaceutical manufacturing, control processes where cleaning is essential, trace chemical identification for antiterrorism and drug detection, and many more. Processes of most interest eliminate contact with contaminants though that may not be required in all embodiments, operate and produce results rapidly, involve little manpower, and are hopefully traceable to standards of chemical identity and concentration. These new methods are focused on optical detection methods from the deep ultraviolet to the infrared.
A problem with any method of contamination concentration detection or determination is certification of accuracy. Whether the method is swabbing, optical detection, or another method, samples of different, known concentrations need to be generated and tested to certify the accuracy of whatever method is used. Ideally, measuring of samples of known concentration for calibration purposes would be in similar form to that of the unknown samples but this is not always the case. Sometimes, surface concentration values are inferred from readings of unknown volume concentrations of samples dissolved in solvents. Furthermore, concentrations of the known samples that have been applied to surfaces and dried have had their concentrations determined by a two-step destructive process. In such processes, a sample is first deposited onto a surface, then the sample is swabbed off the surface over a known area and analyzed via a quantifying methodology such as HPLC, where the areal concentration is determined to be the mass of the material found divided by the known area of sampling. This means that no precise calibration of a spectral analysis tool can be obtained until after measurements of the samples are made with the spectral analysis tool and the samples destroyed to provide precise quantification whereby further use of the sample to confirm continued calibration is not possible as the sample no longer exists. A more satisfactory, efficient, and timely method to achieve spectral instrument calibration is needed.
Accurate, traceable, calibration has remained elusive as uniformly deposited patterns with an a priori quantity of targeted chemicals has remained elusive. Either known quantities were obtainable without adequate uniformity or adequate uniformity could be obtained without precise knowledge of the quantity of material present. The simultaneous lack of both parameters has resulted in a failure to provide calibration samples of known surface concentration which can be used to provide direct accurate conversions between spectral signal measurements to concentration levels of materials of interest. Use of micropipettors have yielded depositions having well known sample quantities that have lacked needed uniformity. Use of spin coating has yielded uniform depositions but without adequate quantification of sample amount. Use of vapor deposition has yielded acceptable uniformity but without adequate quantification. Use of inkjet and piezo printing has also led to uniformity without adequate quantification. Inkjet printing can yield excellent XY printing resolution and accuracy when printed on absorbent surfaces with adequate uniformity of droplet placement. Yet, this technique and piezo printers have droplet sizes that are affected by many physical parameters and thus droplet volumes are not well defined and thus quantity of material deposited is not well defined. For example, surface tension and size of the droplet shot out by the printhead are inversely proportional. Surface tension of the solution is governed by nearly all of the parameters within the solution, solvent type, solute, solute concentration, temperature, and trace contaminants. Because these droplet sizes vary while an area that the printer prints on remains constant, the concentration per unit area changes. Additionally, there are other parameters that change the droplet size, for example the headspace of the sample in the printer cartridge effects the sizes of the deposits. Usually in the beginning when the cartridge is full, the system makes larger deposits, and when the system is less full, the deposits are smaller. Even if a user finds the solvent and solute ratio and verifies the settings on one day, then prints on another day, temperature and humidity changes may cause the system to introduce excess variation into the deposited volume and thus the deposited amount.
A need exists for methods and instruments for providing a plurality of calibration articles of adequate size and known concentration via either completely uniform distribution of material or of sufficiently uniform distributions of material, for example by depositing a plurality of spaced or overlapping droplets of known size having known volume concentrations of sample material such that the reading of articles provides calibration data at a number of different surfaces concentrations so that calibration curves can be created and used to provide quantitative determination of surface concentrations of materials of interest for a given spectral instrument when investigating regions of unknown surface concentration.